1. Field of the Invention
The invention relates to monoclonal antibodies capable of binding to transferrin receptor (TR) cytoplasmic domain, hybridomas producing these antibodies, and methods of using these monoclonal antibodies to isolate endosomal TR cytoplasmic domain sequences.
2. Related Art
Receptor-mediated endocytosis is the mechanism by which a variety of nutrients, hormones, and growth factors are specifically and efficiently transported into the cell. The process of receptor mediated endocytosis is complex and involves several distinct biochemical steps. Typically, the process proceeds by: (1) recruitment of soluble coat proteins to the cell membrane and nucleation of coated pit formation; (2) assembly of coat constituents and growth of the coated pit; (3) acquisition of specific receptors into the growing coated pit; (4) invagination of the cell membrane, (5) coat closure; and (6) membrane fusion wherein the coated pit buds in and pinches off to form a coated vesicle. The contents of the vesicles are ultimately delivered to the endosomes. Key to the entire process of receptor-mediator endocytosis is the internalization signal present in the cytoplasmic tail of the receptor. The cytoplasmic tail interacts with soluble coat proteins during formation of a coated pit.
Receptors such as the transferrin receptor (TR) and the low-density lipoprotein (LDL) receptor are constitutively clustered in coated pits and undergo rapid internalization in both the presence and absence of ligand. Other receptors such as epidermal growth factor (EGF) receptor are only concentrated in coated pits and internalized after binding ligand. Previous studies have established that there are internalization signals in the cytoplasmic domains of constitutively recycling receptors that are believed to interact with adaptor proteins of coated pits and promote high-efficiency endocytosis.
Transferrin receptors (TR) bind the serum transport protein transferrin (Tf) and mediate uptake of iron into the cell. The TR is a homodimeric type II membrane protein consisting of two identical .about.95 kD subunits covalently linked by two intermolecular disulfide bonds (Jing, et al., EMBO J., 6:327-331, 1987). The primary structures of human, mouse, and chicken TRs have been deduced from sequencing of their respective cDNAs and are similar (Schneider, et al., Nature, 311:675-678, 1984; McClelland, et al., Cell, 39:267-274, 1984; Gerhardt, et al., Gene, 102: 249-254, 1991). The human TR has an extracellular domain of 671 amino acids, a single 28-residue transmembrane region and a 61 residue amino-terminal cytoplasmic domain. The TR is a member of the class of ligand transport receptors that are constitutively clustered in coated pits and are rapidly internalized (Goldstein, et al., Ann. Rev. Cell. Biol., 1:1-39, 1985; Trowbridge, Current Opinions in Cell Biology, 3:633-642, 1991). A tetrapeptide sequence, YXRF, in the cytoplasmic tail of the TR has been identified as the recognition motif for high-efficiency endocytosis (Collawn, et al., Cell, 63:1061-1072, 1990).
MAbs have been obtained that react with the external domains of human, mouse, rat and chick TRs (Trowbridge, et al., Proc. Natl. Acad. Sci. USA, 78:3039-3043, 1981; Trowbridge, et al., J. Cell. Physiol., 112:403-410, 1982; Jeffries, et al., Immunology, 54:333-341-1985; Schmidt, et al., Biochem. J., 232:735-741, 1985) and have been useful in characterization of the receptor and studies of its function (Omary, et al., J. Biol. Chem., 256:12888-12892, 1981; Schneider, et al., J. Biol. Chem., 257:8516-8522, 1982; Hopkins, et al., J. Cell Biol., 97:508-521, 1983). Some anti-TR external domain MAbs, either singularly or in combination, block Tf-mediated iron uptake and, as a consequence, inhibit cell growth (Trowbridge, et al., Proc. Natl. Acad. Sci. USA, 79:1175-1179, 1982; Lesley, et al., Mol. Cell. Biol., 4:1675-1681, 1984; White, et al., Cancer Res., 50:6295-6301, 1990). Such antibodies are potential therapeutic agents in the treatment of cancer (White, ibid; Trowbridge, Progress in Allergy, 45:121-146, 1988). However, although MAbs have been produced against the external domain of the TR, no MAbs to the TR cytoplasmic domain have been reported even though such MAbs would be useful in studying the mechanisms associated with vesicle formation and in providing a means for isolating and purifying endosomal vesicles. The present invention addresses this need and provides methods for isolating endosomes by means of antibodies provided by the invention.